Previous studies have demonstrated that nerve cells differentiated from adipose-derived stromal cells after chemical induction have reduced viability; however, the underlying mechanisms remained unclear. In this study, we induced the differentiation of adult adipose-derived stromal cells into astrocytes using chemical induction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry showed that, with increasing induction time, the apoptotic rate gradually increased, and the number of living cells gradually decreased. Immunohistochemical staining demonstrated that the number of glial fibrillary acidic protein-, caspase-3- and caspase-9-positive cells gradually increased with increasing induction time. Transmission electron microscopy revealed typical signs of apoptosis after differentiation. Taken together, our results indicate that caspase-dependent apoptosis is an obstacle to the differentiation of adipose-derived stromal cells into astrocytes. Inhibiting apoptosis may be an important strategy for increasing the efficiency of induction.

Acknowledgments:The author would like to thank Yang XL and Bo Y of Cosmetic Plastic Surgery Center from Kailuan General Hospital in China. Constant supports and technical assistance have been offered generously by teachers from the Experimental Center of Hebei United University in China.Author contributions:Yuan XD was responsible for writing the manuscript, study design, concept, implementation and data arrangement. Sun QY, Ou Y and Zhang LL performed the whole experiment. Wang SJ and Deng HL assisted in data collection and analysis. Zhang WL was responsible for images analysis of electron microscopy. Wu XY provided instruments, equipment and reagents. All authors approved the final version of the paper.Peer review:This study found that caspase-dependent apoptosis is a reason for the death of astrocytes differentiated from adipose-derived stromal cells. The inhibition of this kind of apoptosis may be an important measure to promote the induction effects of astrocytes. This provides a substance basis for treatment and repair of brain injury and neurodegenerative disease using astrocytes.

Volunteers were 13 healthy adults, aged 20-35 years, from the Physical Check-up Center, Kailuan General Hospital, Tangshan, Hebei Province, China. Needle aspiration was used to extract abdominal subcutaneous adipose tissue of adult volunteers without endocrine or hematological diseases. 10-30 mL adipose tissue was collected each time. Written informed consent from the volunteers was obtained. The protocol was approved by the Medical Ethics Committee of Kailuan General Hospital of Hebei United University, China.

Based on the method of Ye et al. (2010) and other experimental protocols, 0.1% collagenase type I (Solarbio, Beijing, China) was added, and the tissue was placed in a 37°C water bath for digestion for 1 hour and then centrifuged at 1,000 r/min for 5 minutes. Supernatant was aspirated out. The undigested tissues and the underlying cells were stirred to mix, and then filtered through a 100-mesh sieve. The samples were centrifuged at 1,000 r/min for 5 minutes, and the supernatant was removed. The remaining cell pellet was seeded into culture flasks at a density of 8 × 10 3 /cm 2 and placed in a 37°C, 5% CO 2 humidified incubator. The culture medium was replaced after 48 hours to remove residual erythrocytes and non-adherent impurities. The culture medium was replaced every 2-3 days. About 10-14 days after the cells reached 90% confluence, trypsin-ethylenediaminetetraacetic acid was used for digestion, and cells were passaged at a 1:2 ratio. Morphological changes in cells were observed using an inverted phase contrast microscope (Olympus, Tokyo, Japan).

Adipose-derived stromal cells at passages 3-6 were digested and seeded onto 12-well culture plates, and inoculated at a density of 1 × 10 5 cells/well. At 48 hours, and 7, 14 and 21 days after induction, cells in each well were incubated with 5 mg/mL MTT (Sigma), 100 μL, at 37°C for 4 hours. The liquid in the well was discarded, and 1,000 μL dimethyl sulfoxide was added to each well. The plates were then agitated at a low speed of 1,000 r/min for 15 minutes. 100 μL aliquots of the solutions were transferred to a 96-well plate and the absorbance at 490 nm was measured using a microplate reader (Thermo Scientific, Pittsburgh, PA, USA). The absorbance is directly proportional to the number of living cells (Wyllie, 1980). Each experiment was repeated five times.

Quantification of early apoptotic cells by flow cytometry

The cells induced for 48 hours, and 7, 14 and 21 days were digested and collected. A single cell suspension was prepared at a concentration of 1 × 10 6 /mL and centrifuged at 1,000 r/min for 5 minutes. The supernatant was discarded, and then 190 μL buffer (Invitrogen, Carlsbad, CA, USA) and 10 μL PI dye solution (Invitrogen) were added. The samples were protected from light and quantified by flow cytometry (BD FACSCalibur, San Jose, CA, USA) within 1 hour. Each experiment was repeated three times.

Ultrastructural characteristics of astrocytes and apoptotic cells after induction

The cells induced for 14 days were digested, centrifuged, and fixed in 3% glutaraldehyde and 1% osmic acid, followed by propionaldehyde dehydration and epoxy resin embedding. Cells were sliced using a microtome (Abnova, Walnut, CA, USA) and stained with 2% uranyl acetate and lead citrate. The ultrastructure of cells was observed and photographed with a transmission electron microscope (H7650, Hitachi, Tokyo, Japan).

Statistical analysis

All experimental data were analyzed using SPSS 13.0 (SPSS, Chicago, IL, USA). Measurement data were expressed as mean ± SD. Intragroup differences were compared using one-way analysis of variance followed by Student-Newman-Keuls test. Values of P < 0.05 were considered statistically significant.

Primary cultured adipose-derived stromal cells adhered at 24 hours, and were observed under the inverted phase contrast microscope. Cells had a triangular and short fusiform appearance [Figure 1]A. After 48 hours of culture, the cells had a long fusiform shape, similar to that of the fibroblasts. At 7-10 days, a large number of long spindle-shaped cells were arranged in a whorl pattern [Figure 1]B. When passaged to the third generation, adipose-derived stromal cells were almost fusiform. At 24 hours after induction, the cytoplasm retracted to the nucleus [Figure 1]C. At 48 hours after induction, round, polygonal or irregular-shaped cells were surrounded by a halo. Parts of cell bodies stretched out slender processes and multiple branches. The cytoplasm was uniform, the nucleus was oval or round, and nucleoli were visible. At 7 days after induction, some of the cells showed the shape of typical astrocytes. Simultaneously, cell protrusions were more extensive, slender branches increased in number and displayed a reticular appearance. The cytoplasm was uniform, the nucleus was large, oval or round, and more biased towards one side of the cell body. Nucleoli were clearly visible [Figure 1]D. At 14 days after induction [Figure 1]E, cell morphology showed no significant changes from that at 7 days. At 21 days after induction, the number of cells was significantly reduced [Figure 1]F. Cells were triangular or irregularly shaped, and cell protrusions were shorter and less numerous than at 14 days of induction. The nucleus showed no significant changes compared with day 14.

Figure 1: Changes in cell morphology before and after ADSC induction (inverted phase contrast microscope).(A) Primary ADSCs cultured for 24 hours. Cells (arrow) are triangular (× 100). (B) ADSCs at the third passage at 7 days. Cells were fusiform and formed a whorl pattern (× 40). (C) At 48 hours, cell bodies of ADSCs (arrow) were polygonal, with a surrounding halo. The nucleus was large and oval-shaped, and slender protrusions with many branches extended out (× 100). (D) At 7 days, refractivity of cell bodies was further enhanced, and there were many slender protrusions around cell bodies, forming a meshwork (× 100). (E) At 14 days, cell morphology showed no significant changes compared with 7 days (× 100). (F) At 21 days, cell processes became shorter and fewer (× 100). ADSCs: Adipose-derived stromal cells.

At 48 hours of induction, glial fibrillary acidic protein expression was observed in a part of the cytoplasm by immunocytochemical staining. The number of glial fibrillary acidic protein-positive cells gradually increased (P < 0.05), and peaked at 14 days. At 21 days, there was no significant difference in the number of glial fibrillary acidic protein-positive cells compared with 14 days (P > 0.05). Expression of caspase-3 and caspase-9 was mainly observed in the cytoplasm. The number of caspase-3- and caspase-9-positive cells gradually increased over the induction period (P < 0.05; [Figure 2], [Figure 3]).

Figure 5 Quantitative distribution of early apoptosis of Adipose-derived stromal cells at various time points after differentiation, as detected by flow cytometry.(A) At 48 hours, values in the upper left quadrant, upper right quadrant, lower left quadrant and lower right quadrant were 3.55%, 8.55%, 83.29% and 4.62%, respectively. (B) At 7 days, values in the upper left quadrant, upper right quadrant, lower left quadrant and lower right quadrant were 4.39%, 10.79%, 77.08% and 7.74%, respectively. (C) At 14 days, values in the upper left quadrant, upper right quadrant, lower left quadrant and lower right quadrant were 6.65%, 8.66%, 72.69% and 12.01%, respectively. (D) At 21 days, values in the upper left quadrant, upper right quadrant, lower left quadrant and lower right quadrant were 4.98%, 10.85%, 69.86% and 14.32%, respectively. The upper left quad­rant: Annexin V−PI+ representing mechanically damaged cells. The upper right quadrant: Annexin V+PI+ representing late apoptotic or necrotic cells. Lower left quadrant: Annexin V−PI− representing living cells. Lower right quadrant: Annexin V+PI− representing early apoptotic cells.

Apoptosis and ultrastructural features of astrocytes differentiated from adipose-derived stromal cells

Under the transmission electron microscope, at 14 days after differentiation, the cell bodies of astrocytes were round or oval. The membrane surface was not smooth, and there were numerous processes. There were varying amounts of bundles of fibers, endoplasmic reticulum, lysosomes and mitochondria in the cytoplasm. Mitochondrial volume was large, and nuclei were oval or irregularly shaped, having 1 or 2 nucleoli, more euchromatin, and reduced and dispersed heterochromatin. The cell bodies of apoptotic astrocytes narrowed, processes from the cell membrane were noticeably decreased or missing, and the surface was smooth. In the cytoplasm, mitochondria exhibited swelling, volume was increased, and the cristae were ruptured and vacuolated. Nuclei were irregular; the nuclear membranes were wrinkled and retracted or even showed karyolysis. Chromatin was reduced, but the amount of heterochromatin was increased. The nuclear chromatin was condensed, and often accumulated near the edges of the nuclear membrane, with blocky or crescent-shaped bodies [Figure 6].

Numerous studies have shown that adipose-derived stromal cells can successfully differentiate into astrocytes in vitro (Safford et al., 2002; Ashjian et al., 2003; Liu et al., 2010; Ou et al., 2011b). The induced cells have the typical morphology of astrocytes and express specific markers, and have a characteristic ultrastructure and electrophysiological properties (Ou et al., 2011a). However, our previous experiments showed that many cells died during adipose-derived stromal cell differentiation, and the number of viable cells decreased with increasing length of induction (Liu et al., 2010; Ou et al., 2011b), thereby limiting further research and hindering the application of astrocytes differentiated from adipose-derived stromal cells. Indeed, in the present study, although the expression rate of glial fibrillary acidic protein, the specific marker of astrocytes, reached a peak of 79.2% at 14 days, the number of viable cells had decreased. Therefore, it is necessary to perform further research on the mechanisms of cell death during the differentiation of adipose-derived stromal cells into astrocytes, and to optimize cell differentiation protocols to improve the survival rate of astrocytes differentiated from adipose-derived stromal cells.

Apoptosis is a process of programmed cell death (Wyllie, 1980; Johansson et al., 2010; Giansanti et al., 2011; Freire, 2012; Igder et al., 2013; Tognon et al., 2013). Our previous studies showed that apoptosis is a major cause of the death of neurons differentiated from adipose-derived stromal cells (Cai et al., 2011; Lu et al., 2012). As the preferred method for early detection of apoptotic cells, Annexin V/PI double-staining and flow cytometry can be used to analyze early apoptotic cells efficiently (Nicoletti et al., 1991; Grebeňová et al., 2004; Samsel et al., 2004; Suárez et al., 2004; Guo et al., 2011; Wlodkowic et al., 2013). Therefore, we studied cell death rate in the early stage by flow cytometry. The results demonstrated that the death rate increased over time, peaking at 21 days of induction. This further confirmed that apoptosis is a major cause of cell death.

Apoptosis includes caspase-dependent apoptosis and caspase-independent apoptosis (Liu et al., 2013). In caspase-dependent apoptosis, the caspase family is a key mediator of cell death. As the preferred method for detecting apoptosis (Zhou et al., 2012), transmission electron microscopy plays a crucial role in its detection (Ji et al., 2011; Würstle et al., 2012). Our findings revealed that caspase-dependent apoptosis is a major apoptotic pathway during cell differentiation. Furthermore, the numbers of caspase-9- and caspase-3-positive cells increased over time and were associated with apoptosis of astrocytes differentiated from adipose-derived stromal cells. The expression rate of caspase-9, the main initiation factor of apoptosis (Fombonne et al., 2012; White et al., 2012; Brentnall et al., 2013; Qin et al., 2013), reached 30.27% at 21 days of induction. However, for caspase-3, the rate was 46.47%, significantly higher than that for caspase-9, suggesting a significant amplification effect. For both caspase-3 and caspase-9, expression is mainly in the cytoplasm, especially around the nucleus; however, there is no significant expression in the protrusions of the induced cells. The expression of caspase-9 was less than the expression of executioner caspase-3, suggesting an amplification effect in the process of induction.

During the differentiation of adipose-derived stromal cells into astrocytes, expression of glial fibrillary acidic protein, an important marker of astrocytes (Bernal and Peterson, 2011; Sokolowski et al., 2011; Yeh et al., 2011; Sukumari-Ramesh et al., 2012; Hoppe et al., 2013), is evenly detected in the cytoplasm as well as in the protrusions. Glial fibrillary acidic protein bundles are evenly distributed in the cytoplasm as well as in the cell processes of normal astrocytes differentiated from adipose-derived stromal cells. More organelles are observed, including mitochondria, endoplasmic reticulum and lysosomes. There is more euchromatin and less heterochromatin in nuclei, and the heterochromatin is dispersed. In contrast, in the cytoplasm of apoptotic cells, the mitochondria, concentrated around the nuclei, were significantly swollen, the cristae were ruptured, and vacuolization was observed. The membranes of nuclei were wrinkled, and some were fragmented. The chromatin was condensed and distributed at the membrane, with a block or crescent shape, typical of apoptosis. Finally, cell morphological changes, including shrinkage, and a decrease or even disappearance of protrusions and microvilli occur.

In summary, adipose-derived stromal cells can differentiate into astrocytes using a chemical inducer mainly consisting of 3-isobutyl-1-methylxanthine. The number of living cells decreases over the duration of induction. Caspase-dependent apoptosis is the main pathway of apoptosis during induction. [59]

Würstle ML, Laussmann MA, Rehm M (2012) The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp Cell Res 318:1213-1220.